Effects Of Heat Stress And Oxidative Stress On Metabolism Of Skeletal Muscle And Protection Of Lipoic Acid In Finishing Pigs

Heat stress and oxidative stress are most common stresses facing the pig production. Both stresses have a detrimental effect on growth performance and meat quality in finishing pigs. Firstly, model on finishing pigs exposure to constant heat stress was established to investigate the effects of heat stress on energy metabolism and proteome of Longissimus dorsi muscle. Secondly, we studied the influences of heat stress and restricted feed intake on the hepatic proteome and antioxidant capacity in finishing pigs. In addition, we established oxidative stress models in vitro and in vivo to investigate the underlying mechanisms where lipoic acid（LA） could alleviate oxidative stress in finishing pigs and decline in meat quality. For these objectives, five experiments were conducted and main results were listed as follows:Exp. 1 Effects of heat stress on energy metabolism and proteome of Longissimus dorsi muscle.24 castrated male crossbreeds between Landrace × Yorkshire sows and Duroc boars（79.00 ± 1.50 kg body weight） were exposed to thermal neutral（TN, 22°C, n=8） ad libitum, heat stress（HS, 30°C, n=8） and ad libitum feeding and ambient temperature at 22°C and pair-fed to the HS diets（pair-fed, PF, n=8） for 3 weeks.HS significantly increased the level of lactic acid（P = 0.02）, activity of hexokinase and pyruvate kinase（P < 0.01） and expression of AMP-activated protein kinas（p-AMPK）（P = 0.02）. Heat stress changed the proteome of Longissimus dorsi muscle. A total of 30 differentially expressed proteins were identified, of which 15 were down-regulated and another 15 were up-regulated. These proteins were involved in carbohydrate metabolism, myofibrillar and cytoskeleton structure, stress response, antioxidant and detoxification, calcium binding and cellular apoptosis. These results indicated that the heat exposure changed intracellular energy metabolism, where the AMP-activated protein kinase was activated. As a result, lactic acid level increased. Moreover, heat stress affected the programmed cell death and degradation of myofibrillar proteins, in turn, suppressed tenderization and meat quality.Exp. 2 Effects of heat stress and reduced feed intake on antioxidant ability and proteome of liver in finishing pigsBased on the heat stress model established in the first experiment, we investigated the effects of heat stress and reduced feed intake on antioxidant ability and proteome of liver in finishing pigs. Heat stress and restricted feed intake significantly reduced liver weight（P < 0.05）, changed activity of glutathione antioxidant system and the hepatic proteome. A total of 45 differentially expressed proteins were identified by MS. GO and KEGG enrichment analysis of these proteins were performed, showing that these proteins were mainly involved in heat shock response and immune defense, oxidative stress response and cellular apoptosis.These results demonstrated that Heat-load, independent of reduced feed intake, induced an innate immune response, while food restriction caused stress and cellular apoptosis. To neutralize the effects of chronic HS, the liver responds by up-regulating HSPs to maintain protein folding structures, enhancing the anti-oxidant pathway for a balanced redox state, and over-expression of the proteasome for thedegradation of oxidized or misfolded proteins.Exp. 3 Putative signal pathways: protection of lipoic acid（LA） against H₂O₂-induced oxidative stress in C2C12 cells.The present trial was conducted to study the effects of LA on cell proliferation, antioxidant enzymes activity, and expression of Nrf2, p-MAPK in C2C12 cells under oxidative stress induced by H₂O₂. Based on the preliminary experiment, the oxidative stress C2C12 model was established according to the condition: the concentration of medium H₂O₂ is 0.4 m M and 0.8 m M, the time is 0.5 h and LA is 0 m M 、0.2 m M、0.4 m M、0.8 m M and 1.2 m M. The results showed that LA significantly increased rate of proliferation, activities of SOD and GSH level, and expression of p-MAPK and Nrf2（P < 0.01） under 0.4m M H₂O₂-induced oxidative stress. Those data indicated that LA may relieve oxidative stress by increasing the synthesis of antioxidant enzymes mediated by p-MAPK and Nrf2.Exp. 4 Effects of LA on growth performance and serum antioxidant enzyme activity of finishing pigs subjected to oxidative stressTo gain insights into the effects of LA on growth performance and serum antioxidant enzyme activity of finishing pigs under oxidative stress, an oxidative stress model was established by intraperitoneal injection with diquat of finishing pigs. Twenty four larger white finishing pigs were randomly allocated to 2 groups of 12 pigs per group. These pigs were fed with formulated diet containing 0 and 800 mg/kg LA, respectively. After 14 d, one half of pigs in each group were challenged with 8 mg/kg WB diquat, the other was injected with the equal physiological saline. Finishing pigs were housed in single cage, and the whole experiment lasted 29 d.The results showed that diquat compromised growth performance, including significantly reduced ADG, ADFI and increased F/G（P < 0.01）. In consistent with them, diquat significantly reduced the serum antioxidant enzyme activity（P < 0.01）. Under physiological condition, all of these parameters were not changed significantly（P > 0.05）. However, diary supplement LA can relieve the decline in ADFI, ADG and SOD activity under oxidative stress. These data indicated that finishing pigs challenged with 8mg/kg BW diquat display reduced growth performance, lower serum antioxidant enzyme activity, suggesting that finishing pigs entered the oxidative stress. Moreover, diary supplement LA can mitigate the negative effects of diquat-induced oxidative stress.Exp. 5 Underlying mechanisms: protective effects of LA on finishing pigs subjected to oxidative stress Based on the results from experiment 3 and 4, the present study was conducted to investigate the underlying mechanism where LA can mitigate negative effects of oxidative stress on muscle and meat quality in finishing pigs. The main results are as follows:pH24 h at 24 h post-mortem was significantly reduced under oxidative stress induced by diquat（P < 0.05）, however, diary supplement LA could significantly increase the redness（a45min） at 45 min post-mortem and yellowness（b24 h） and shear force at 24 h post-mortem（P < 0.01）. SOD activity and protein and m RNA expression of SOD were significantly induced by Diquat injection, and diary supplement LA increased the m RNA expression of SOD（P < 0.05）. Diquat injection significantly decreased theexpression of p-p38（P < 0.05） and increased the expression of Nrf2（P < 0.05）. Feeding LA-supplemented diets significantly decreased the expression of Nrf2（P < 0.05）.In conclusion, heat stress changes intracellular energy metabolism of Longissimus dorsi muscle, activating AMPK pathway, in turn, increasing glycolysis. As a result, lactic acid level increases, which affects meat quality. In addition, heat stress affects the programmed cell death and degradation of myofibrillar proteins, and suppresses the tenderization and meat quality. Heat stress and feed restriction change activity of glutathione antioxidant system and liver proteome. Heat load, independent of reduced feed intake, triggers an innate immune response. Heat stress results in oxidative stress response while both stresses can induce cellular apoptosis.Oxidative stress reduced growth performance, serum antioxidant enzyme activity while diary supplement LA could mitigate growth restriction and promote antioxidant ability of finishing pigs. LA may relieve oxidative stress by increasing the synthesis of antioxidant enzymes mediated by p-MAPK and Nrf2 from both in vitro and in vivo experiment.